The complex group of opioid receptors is biologically and clinically important in mediating the action of endogenous and exogenous opioids. Despite the clinical advantages of developing drugs which serve as specific agonists and antagonists for opioid receptor subtypes, studies to date have been limited by the inability to satisfactorily isolate and characterize these multiple opioid receptors. This project is aimed at better defining the biochemical and molecular basis of opioid receptor system and actions. The opioid receptor system is believed to consist of, at least, an opioid ligand binding receptor protein and a guanine nucleotide regulatory protein (G-protein) that regulates the receptor affinity and serves as a signal transducer to produce cellular biochemical responses such as inhibition of adenylate cyclase or neurotransmitter release. Based on this model the plan is to define the role of G-protein in the opioid receptor system Substantial quantity of G-protein can be isolated from rat brain and lung tissues. Opioid receptor has been solubilized with high yield and stability. This proposal is to reconstitute G-protein back to membranes containing "uncoupled" opioid receptor, to use G-protein as a tool to purifiy solubilized opioid receptors to homogeneity, and to study possible mechanisms of opioid tolerance that involves G-protein. A rat spinal tolerance model was established which one can manipulate to achieve selective as weH as cross tolerance to specific mu- and delta-agonosts. Using this model it has been shown that alternated mu- and delta- therapy can reduce the degree of tolerance. It was also demostrated in the rat spinal model that tolerance preceded receptor down regulation. This proposal is to study the mu- and delta-receptor binding and opioid sensitive GTPase activities in the spinal cord membranes to correlate with the development of opioid tolerance. The spinal membranes from tolerant animals will also be used in reconstitution experiments to study the role of G-proteins in opioid tolerance. Information generated from this proposal will aid in the elucidation of the molecular basis of thc opioid receptor system and the mechanism of opioid actions.